| Abstract |
Butylated hydroxyanisole (BHA) is an antioxidant and class-2B carcinogen. It is biotransformed to tert-butylhydroquinone (TBHQ), which readily auto-oxidizes to the electrophilic metabolite tert-butylbenzoquinone (TBQ). BHA and TBHQ activate Nrf2, a transcription factor that is negatively regulated by Keap1 and plays a role in the initial response to chemicals causing oxidative or electrophilic stress, although, the exact mechanism of Nrf2 activation remains unclear. Here, we examined the role of TBQ in Nrf2 activation. Exposure of RAW264.7 cells to TBQ activated Nrf2 and up-regulated its downstream proteins; under these conditions, TBQ produced cellular reactive oxygen species (ROS). However, while pretreatment with catalase conjugated with polyethylene glycol (PEG-CAT) did not affect the TBQ-induced activation of Nrf2, the ROS generation caused by TBQ was entirely abolished by PEG-CAT, suggesting that ROS is not the dominant factor for TBQ-dependent Nrf2 activation. A click chemistry technique indicated that TBQ chemically modifies Keap1. Furthermore, ultrahigh performance liquid chromatography-tandem mass spectrometry analysis with purified Keap1 revealed that TBQ covalently binds to Keap1 through Cys23, Cys151, Cys226, and Cys368. These results suggest that TBQ derived from BHA activates Nrf2 through electrophilic modification of Keap1 rather than ROS formation.
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